Optimal concentration of p-aminobenzoic acid and folic acid in the in vitro assay of antifolates against Plasmodium falciparum

In vitro tests for Plasmodium falciparum sensitivity to pyrimethamine, sulfadoxine, and both drugs in combination were performed in four kinds of culture medium, each differing in p-amino benzoic acid (PABA) and folic acid concentrations. Results of the tests using pyrimethamine-sensitive and pyrimethamine-resistant isolates indicated that drug activity was reduced proportionally to the concentrations of these two growth factors in the medium. The optimal concentrations of PABA and folic acid for parasite growth and drug susceptibility, as evaluated by microscopic examination and by the extent of incorporation of radioactive 14C-pyrimethamine and 14C-sulfadoxine, were 10 ng/ml and 2 ng/ml, respectively. Depletion of PABA and folic acid from the medium had no effect on drug-resistant parasites but multiplication of drug-sensitive isolates was markedly reduced. Medium containing 0.5 ng/ml PABA and 10 ng/ml folic acid was the best for parasite growth regardless of the degree of drug sensitivity. Results obtained by using this medium agreed most closely with results from in vivo observations.     

The effect of 5-fluorouracil and 5-fluorocytosine on the development of the filarial nematodes Brugia pahangi and Dirofilaria immitis

5-fluorouracil and 5-fluorodeoxyuridine at 30 mg/kg body weight daily for four days inhibit microfilarial production in Brugia pahangi in the jird. Disruption of intrauterine embryogenesis was observed in treated female worms but the compounds were not macrofilaricidal or microfilaricidal under the conditions employed. 5-fluorocytosine possessed no filaricidal or embryostatic activity. The inhibition of microfilaria production by 5-fluorouracil was temporary and larval production was resumed within nine weeks. The compound also inhibited the development of B. pahangi and Dirofilaria immitis larvae in the mosquito Aedes aegypti, when administered to cages of mosquitoes as a 0.01 or 0.001% solution in a 10% aqueous sucrose solution on cotton wool wicks. The development of infective larvae of B. pahangi in the jird was inhibited by 5-fluorouracil.     

A preliminary study on in vitro transmission of Dirofilaria immitis infective stage larvae by Aedes aegypti (L.) (Diptera: Culicidae)

This study was performed to study an in vitro transmission of infective stage larvae from the mosquito proboscis. There were five experiments with 949 mosquitoes. Liverpool strain of Aedes aegypti (L.) were used in this study. They were allowed to feed on D. immitis infected dogs with different microfilarial levels which were 1,650, 1,950, 9,000, 9,250, and 11,550 microfilariae per one ml of blood. Mosquitoes were forced to feed on solution (5% sucrose in 5% dog serum) in capillary tubes for 20 minutes at 7-34 days post-blood feeding. Solutions in capillary tubes then were examined and mosquitoes were dissected and examined for D. immitis larvae under a light microscope. Second stage larvae could be found in the abdomen and malpighian tubules of mosquitoes and third stage larvae can be found in the abdomen, malpighian tubules, thorax, and proboscis of mosquitoes with different levels of infection. No larvae were detected in the solution in capillary tubes of all experiments.     

In vitro development of Brugia pahangi and Brugia malayi in cultured mosquito thoraces.

In vitro cultivation of Brugia pahangi and subperiodic Brugia malayi one-day old larvae to infective stage larvae (L3) within thoraces excised from Aedes aegypti (Black Eye, Liverpool) and Anopheles quadrimaculatus was attempted. The mosquito thoraces were excised under aseptic conditions, 24 h after a blood meal on either B. pahangi- or B. malayi-infected jirds. The excised thoraces were washed aseptically and inoculated into a diphasic media. A nutrient agar base was overlaid with either Grace's insect cell culture medium or Schneider's Drosophila medium or a mixture (1:1) of these two media. Each overlay medium contained a 1 x concentration of antibiotic/antimycotic mixture plus 20% fetal bovine serum. The excised thoraces provided the intracellular milieu for development of Brugia larvae. In Grace's or Schneider's insect tissue culture medium alone, the filaria larvae of both species developed only to the second larval stage after 12 days; whereas, in a mixture (1:1) of Grace's and Schneider's media, some one-day old larvae of both Brugia species developed to the infective larval (L3) stage after 12 days. However, large numbers of both species of larvae developed to the infective larval stage when, prior to providing an infective blood meal, the mosquitoes of both species were fed 1 x concentration of antibiotic/antimycotic mixture in a 10% sucrose solution containing 0.1% p-aminobenzoic acid for 6 days. These results showed for the first time that if one-day old Brugia larvae are confined intracellularly in excised thoraces, they can then develop in insect tissue culture media without adding a feeder layer of mosquito cells or conditioning the media with mosquito cell lines.     

Folic acid malabsorption in atrophic gastritis. Possible compensation by bacterial folate synthesis

Folic acid absorption was studied in 12 elderly subjects with atrophic gastritis and 10 elderly normal controls using tritium-labeled pteroylmonoglutamic acid. Two folic acid absorption tests were carried out on each subject with 120 ml of either water or 0.1 N HCl. Folic acid absorption was significantly lower in subjects with atrophic gastritis than in normal controls (31% vs. 51%, respectively; p less than 0.01). In subjects with atrophic gastritis, folic acid absorption rose significantly to 54% (p less than 0.001) when administered with acid, but did not change in normal controls (50%). Serum folate levels were normal in all subjects. Proximal small intestinal pH was higher in atrophic gastritis subjects than in normal controls (7.1 vs. 6.7, respectively; p less than 0.05), as were bacterial counts of small intestinal fluid (p less than 0.01). Bacteria cultured from the aspirates of subjects with atrophic gastritis were able to synthesize folate in vitro when incubated in a folate-free medium. Atrophic gastritis results in folic acid malabsorption but not in folate deficiency, possibly due to increased bacterial synthesis of folate in the small intestine.     

Laboratory transmission of lymphatic filariasis by vector mosquitoes

Aedes togoi and Ae. aegypti were used to examine the transmission potential of Brugia pahangi to one of its natural hosts, the domestic cat. Although a larger proportion of microfilariae taken in by Ae. togoi developed into infective larvae, the total number of B. pahangi larvae recovered from a cat exposed to Ae. aegypti was larger than from a cat exposed to Ae. togoi. Factors influencing the transmission dynamics included: development of microfilariae to infective larvae; survival of mosquitoes; willingness to take repeated blood meals; and proportion of infective larvae that egress from mosquitoes during the feeding process. From 19 to 25% of infective larvae were transferred to a susceptible host. The feasibility of using a Brugia-cat model to do comparative vector efficiency studies was demonstrated.     

Quantitative aspects of the development of mosquito transmitted Brugia malayi and Brugia pahangi and their distribution in Jirds, Meriones unguiculatus.

Twenty-two jirds, Meriones unguiculatus, were exposed to the bites of 2250 females of Aedes aegypti carrying an estimated total of 2464 larvae of Brugia malayi, and 13 jirds were offered for blood feeding to 1450 mosquitoes infected with about 4460 larvae of Brugia pahangi. On necropsy of the jirds, four months after feeding of the mosquitoes, a total of 88 adult filariae of B. malayi and 143 of B. pahangi were recovered in 20 and 13 jirds respectively. The majority of the adult filariae was obtained from the testes (48,9% of B. malayi and 53,2% of B. pahangi), from the heart-lung-system (26,1% and 45,6%), and additional in B. malayi from the tail (19,3%). It can be estimated that 3,6% (3,2%) of all infective larvae of B. malayi (B. pahangi), carried by Aedes aegypti females before feeding, reached maturity in the jird host after they had left the vectors during the blood meal. Microfilariae were found in the peripheral blood in seven of B. malayi infected jirds and in eleven of the jirds infected with B. pahangi.     

Replication of dengue type 2 virus in Culex quinquefasciatus (Diptera: Culicidae)

We were able to infect Culex quinquefasciatus by the parenteral route with dengue virus type 2. The percentage of mosquitoes infected was dose dependent and we obtained a rate of 45.6% infected Cx. quinquefasciatus when a 10(5.9) MID50 (mosquito infectious dose for 50% of the individuals as measured in Aedes aegypti) of dengue virus type 2 per mosquito was used. Infection was detected by an immunofluorescent assay performed on mosquito head squashes 14 days after infection. The replication of dengue virus in Cx. quinquefasciatus was either at a very low level of magnitude or generated a large number of noninfectious particles since the triturated bodies of infected Cx. quinquefasciatus did not infect Ae. aegypti mosquitoes when inoculated parenterally. We were unable to infect Cx. quinquefasciatus females orally with an artificial meal that infected 100% of Ae. aegypti females. These findings lead us to agree with the consensus that Cx. quinquefasciatus should not be considered a biological vector of dengue viruses.     

Vector competence of mosquitoes as a marker to distinguish Central American and Mexican epizootic from enzootic strains of Venezuelan encephalitis virus.

Two epizootic strains of Venezuelan encephalitis (VE) virus from Central America and Mexico were transmitted by a colonized epizootic vector mosquito, Aedes taeniorhynchus, at higher rates than were two enzootic strains when the mosquitoes were infected by intrathroacic inoculation or feeding of virus. Differences in transmission rates also occurred with colonized Aedes aegypti, but were less marked. Following intrathoracic inoculation of A. taeniorhynchus or A. aegypti, epizootic strains grew to slightly higher concentrations in the mosquitoes than did enzootic strains. Intestinal thresholds of infection for A. taeniorhynchus were slightly lower for epizootic than for enzootic virus strains, but were essentially equal for A. aegypti. Only a small percentage of individual Culex pipiens quinquefasciatus mosquitoes supported growth of epizootic VE virus, and only 1 of 6 tested C. p. quinquefasciatus transmitted virus by bite. Thus, transmission and growth of virus in these Aedes mosquitoes distinguished between these epizootic and enzootic strains of VE virus.     

The susceptibility of five laboratory colonies of mosquitoes to the human nematode Wuchereria bancrofti (Cobbold)

Laboratory colonies of Anopheles gambiae, An. arabiensis, An. merus, An. quadriannulatus and Aedes aegypti formosus were artificially fed on blood containing microfilariae of Wuchereria bancrofti. The anopheline colonies all supported parasite development to the infective stage, with An. quadriannulatus being the most heavily infected. The parasite did not develop at all in the Ae. aegypti formosus colony.     

Effect of temperature on the vector efficiency of Aedes aegypti for dengue 2 virus

The effect of temperature on the ability of Aedes aegypti to transmit dengue (DEN) 2 virus to rhesus monkeys was assessed as a possible explanation for the seasonal variation in the incidence of dengue hemorrhagic fever in Bangkok, Thailand. In two laboratory experiments, a Bangkok strain of Ae. aegypti was allowed to feed upon viremic monkeys infected with DEN-2 virus. Blood-engorged mosquitoes were separated into two groups and retained at constant temperatures. Virus infection and transmission rates were determined for Ae. aegypti at intervals ranging from 4 to 7 days during a 25-day incubation period. Results of the first experiment for mosquitoes infected with a low dose of DEN-2 virus and maintained at 20, 24, 26, and 30 degrees C, indicated that the infection rate ranged from 25% to 75% depending on the incubation period. However, DEN-2 virus was transmitted to monkeys only by Ae. aegypti retained at 30 degrees C for 25 days. In the second experiment, the infection rate for Ae. aegypti that ingested a higher viral dose, and incubated at 26, 30, 32, and 35 degrees C ranged from 67% to 95%. DEN-2 virus was transmitted to monkeys only by mosquitoes maintained at greater than or equal to 30 degrees C. The extrinsic incubation period was 12 days for mosquitoes at 30 degrees C, and was reduced to 7 days for mosquitoes incubated at 32 degrees C and 35 degrees C. These results imply that temperature-induced variations in the vector efficiency of Ae. aegypti may be a significant determinant in the annual cyclic pattern of dengue hemorrhagic fever epidemics in Bangkok.     

Infection of Anopheles darlingi fed on patients with Plasmodium falciparum before and after treatment with quinine or quinine plus tetracycline

Anopheles darlingi fed on eight falciparum malaria patients with gametocytes before and after treatment with quinine sulfate or quinine sulfate plus tetracycline became infected. Quinine and quinine plus tetracycline had no apparent sporontocidal or gametocytocidal effect on late stage immature and mature gametocytes. Plasmodium falciparum gametocytes are persistent and infected mosquitoes for up to 21 days after patients were treated with quinine plus tetracycline. Sporogonic development was similar for groups of mosquitoes fed before and after patients were treated with these schizontocides. The percentages of infected mosquitoes that developed salivary gland infections were also similar for groups of mosquitoes fed before and after treatment. Twenty-four hours after treatment with 45 mg of primaquine phosphate, falciparum malaria patients were not infective to An. darlingi.     

Effect of folic acid supplementation on genomic DNA methylation in patients with colorectal adenoma

BACKGROUND AND AIMS: A low dietary folate intake can cause genomic DNA hypomethylation and may increase the risk of colorectal neoplasia. The hypothesis that folic acid supplementation increases DNA methylation in leucocytes and colorectal mucosa was tested in 31 patients with histologically confirmed colorectal adenoma using a randomised, double blind, placebo controlled, parallel design. METHODS: Subjects were randomised to receive either 400 microg/day folic acid supplement (n = 15) or placebo (n = 16) for 10 weeks. Genomic DNA methylation, serum and erythrocyte folate, and plasma homocysteine concentrations were measured at baseline and post intervention. RESULTS: Folic acid supplementation increased serum and erythrocyte folate concentrations by 81% (95% confidence interval (CI) 57-104%; p<0.001 v placebo) and 57% (95% CI 40-74%; p<0.001 v placebo), respectively, and decreased plasma homocysteine concentration by 12% (95% CI 4-20%; p = 0.01 v placebo). Folic acid supplementation resulted in increases in DNA methylation of 31% (95% CI 16-47%; p = 0.05 v placebo) in leucocytes and 25% (95% CI 11-39%; p = 0.09 v placebo) in colonic mucosa. CONCLUSIONS: These results suggest that DNA hypomethylation can be reversed by physiological intakes of folic acid.     

Growth characteristics of ChimeriVax-DEN2 vaccine virus in Aedes aegypti and Aedes albopictus mosquitoes

The chimeric yellow fever (YF) 17D-dengue type 2 (ChimeriVax-DEN2) vaccine virus developed by Acambis, Inc. (Cambridge, MA) contains the prM and E genes of wild-type (wt) dengue 2 (DEN-2) (strain PUO-218) virus in the YF vaccine virus (strain 17D) backbone. The potential of ChimeriVax-DEN2 virus to infect and be transmitted by Aedes aegypti, the principal DEN and YF virus mosquito vector, and Aedes albopictus, a species that occurs in areas of active transmission of YF and DEN viruses, was evaluated. Mosquitoes were intrathoracically (IT) inoculated with virus or were fed a virus-laden blood meal, and the replication kinetics of ChimeriVax-DEN2 were compared with the wt DEN-2 and YF 17D vaccine viruses. Replication of YF 17D virus is attenuated in cultured Ae. albopictus C6/36 mosquito cells and in Ae. aegypti and Ae. albopictus mosquitoes. Growth of ChimeriVax-DEN2 virus similarly was restricted in C6/36 cells and in mosquitoes. ChimeriVax-DEN2 replicated in 56% of IT inoculated Ae. aegypti, and virus disseminated to head tissue in 36%, with a mean viral titer of 1.8 log10 PFU/mosquito. Of mosquitoes, 16% of Ae. aegypti and 24% of Ae. albopictus were infected 14 days after a blood meal containing ChimeriVax-DEN2, but virus did not disseminate to head tissue. In contrast, DEN-2 replicated in all IT inoculated and orally infected Ae. aegypti (mean titer 5.5 log10 PFU/mosquito), and virus disseminated to head tissue in 95%. Of Ae. albopictus, 84% were infected after a blood meal containing DEN-2 virus; dissemination occurred in 36%. Replication of ChimeriVax-DEN2 virus in mosquitoes corresponded to that of YF 17D vaccine virus, which is restricted in its ability to infect and replicate in mosquitoes. Therefore, transmission of ChimeriVax-DEN2 virus by vector mosquitoes is unlikely.     

Electroantennogram, flight orientation and oviposition responses of Anopheles stephensi and Aedes aegypti to a fatty acid ester-propyl octadecanoate

Studies were carried out to evaluate the role of a C(21)-fatty acid ester; propyl octadecanoate (PO) for olfaction-mediated behavioral responses of urban malaria vector, Anopheles stephensi and dengue vector, Aedes aegypti mosquitoes using electroantennogram (EAG), flight orientation and oviposition experiments. Dose dependent electrophysiological responses were recorded for PO from the antenna of both mosquito species in which 10(-5)g elicited significant EAG response. An. stephensi exhibited 2.4, 4.2 and 5.5 fold increased EAG response compared to control, while Ae. aegypti showed 1.9, 4.6 and 5.8 fold EAG responses respectively at 10(-7)g, 10(-6)g and 10(-5)g doses. In the Y-tube olfactometer, 77-80% gravid females of An. stephensi, and 64-77% of Ae. aegypti were caught in the chambers releasing 10(-6)g and 10(-5)g plume of PO. The synthetic fatty acid ester loaded onto an effervescent tablet at 0.1mg/L, 1mg/L and 10mg/L elicited increased ovipositional responses from gravid mosquitoes compared to control. The oviposition activity indices (OAI) of An. stephensi females were +0.40, +0.51 and +0.58, whereas the OAI for Ae. aegypti females were +0.05, +0.36 and +0.57 respectively in 0.1, 1, 10mg/L of PO; indicated concentration dependent increased egg deposition. Similarly, in the residual activity studies, oviposition substrates treated with PO on effervescent tablet at 1mg/L and 10mg/L received significantly increased egg deposition by gravid females of both mosquito species for up to 1 week compared to control substrates. PO can potentially be used in ovitraps to monitor An. stephensi and Ae. aegypti populations in the vector surveillance programs.     

Persistence of dengue-3 virus through transovarial transmission passage in successive generations of Aedes aegypti mosquitoes

Progeny of Aedes aegypti mosquitoes infected intrathoracically with dengue-3 virus was reared to subsequent generations. In each generation, blood-fed females were confined individually and the eggs obtained from the transovarially infected females were pooled. The seventh generation obtained from the infected parental mosquitoes showed that virus could persist in mosquitoes in successive generations through transovarial passage. The rate of vertical transmission initially increased in the few generations (F1-F2), but in subsequent generations it was found to be steady. Parental mosquitoes inoculated with virus showed higher mortality than the diluent-inoculated controls. There was an increase in the larval duration of transovarially infected batches at the seventh generation when compared with uninfected control mosquitoes. The fecundity and fertility of the transovarially infected batches of mosquitoes was also affected when compared with the controls. This is the first report demonstrating persistence of dengue virus in the successive generations of mosquitoes infected through vertical transmission. These observations, which have great epidemiologic importance, suggest that vector mosquitoes may play an important role in the maintenance of virus in nature, and that mosquitoes may act as reservoirs of these viruses.     

Evaluation of cyfluthrin and fenfluthrin for their insecticidal activity against three vector mosquitoes

The EC50/EC90 concentrations of cyfluthrin and fenfluthrin were tested for their activity against different developmental stages of three important vector mosquitoes viz., Anopheles stephensi Liston, Aedes aegypti (Linn.) and Culex quinquefasciatus Say. The EC90 concentrations of both cyfluthrin and fenfluthrin showed ovicidal effect on An. Stephensi and Ae. aegypti whereas EC90 of cyfluthrin checked the hatching of eggs completely in Cx. quinquefasciatus. Fenfluthrin at EC50 concentration reduced the percentage of hatching significantly (p < 0.05) only in An. stephensi. Both the compounds were more active against the fourth larval instars of all mosquito species and cyfluthrin in culicines (17.3% Ae. aegypti = 9.1%) and fenfluthrin in anophenlines (An. stephensi = 36.8%) brought about maximum inhibition in adult emergence. Various types/degrees of morphogenetic aberrations were induced in all mosquito species on treatment with these compounds. Cyfluthrin treated female mosquitoes showed reduced fecundity rates in An. stephensi (p < 0.05), Cx. quinquefasciatus (p < 0.001) and fenfluthrin treated in An. stephensi (p < 0.5) and Ae. aegypti (p < 0.05). The fertility rates of all the mosquito species were significantly reduced (p < 0.001) by both the compounds.     

Folic acid is positively associated with uteroplacental vascular resistance: The Generation R Study

Background and aims

Periconception folic acid supplementation may influence early placentation processes and thereby the occurrence of hypertensive pregnancy disorders. For this reason we examined the associations between periconception folic acid supplementation and uteroplacental vascular resistance, blood pressure, and the risks of gestational hypertension and preeclampsia, in 5993 pregnant women, participating in a population-based cohort study.

Methods and results

Folic acid supplementation was assessed by questionnaire. Mean pulsatility index (PI) and resistance index (RI) of the uterine (UtA) and umbilical arteries (UmA) were measured by Doppler ultrasound in mid- and late pregnancy. Systolic and diastolic blood pressures (SBP, DBP) were measured in early, mid- and late pregnancy. Compared to women who did not use folic acid, preconception folic acid users had a slightly lower UtA-RI in mid-pregnancy [β −0.02, 95% confidence interval (CI) −0.03, −0.01] and late pregnancy [β −0.02, 95% CI −0.03, −0.001], a lower UtA-PI in mid-pregnancy [β −0.06, 95% CI −0.1, −0.03] and late pregnancy [β −0.03, 95% CI −0.05, −0.01], as well as tendencies towards a lower UmA-PI in mid-pregnancy [β −0.02, 95% CI −0.04, −0.001] and late pregnancy [β −0.01, 95% CI −0.02, 0.01]. Additionally, these women had slightly higher SBP and DBP throughout pregnancy. Neither the patterns of blood-pressure change during pregnancy, nor the risk of gestational hypertension and preeclampsia differed between the folic acid categories.

Conclusion

Periconception folic acid supplementation is associated with lower uteroplacental vascular resistance and higher blood pressures during pregnancy. The effects are small and within physiologic ranges and seem not associated with the risk of hypertensive pregnancy disorders.